Sheep red blood cells armed with anti-CD20 single-chain variable fragments (scFvs) fused to a glycosylphosphatidylinositol (GPI) anchor: a strategy to target CD20-positive tumor cells

Single-chain variable fragment antibodies (scFv) retain antigen specificity and offer advantages over intact antibodies as therapeutic agents. We cloned the cDNA of the VH and Vκ regions from a mouse hybridoma (HB-9645) directed against human CD20. In addition to the basic scFv construct (Vκ-L-VH), we genetically engineered a secretory signal, six histidine residues, and a 'Flu' tag to facilitate secretion, purification, and detection. A glycosyl-phosphatidylinositol (GPI) modification signal was added at the C terminus. The GPI-tagged and the non-tagged scFvs were expressed in high yields on the surface of stably transfected insect cells. The CD20-binding properties of purified non-GPI tagged scFv were examined using flow cytometry and immunocytochemistry. The non-GPI-tagged scFv selectively recognizes CD20-positive cells in a concentration-dependent manner. Double-flow cytometry analysis using fresh peripheral blood lymphocytes and WSU-FSCCL cells revealed that our scFv resolves the B-cell population better than the intact antibody. The GPI-tagged scFv was loaded onto the surface of sheep erythrocytes to form rosettes with CD20-positive cells. The genetically engineered anti-CD20 scFv and GPI-tagged derivative have binding specificity for the CD20 antigen. The scFvs described here has potential uses as an in vivo tumor-imaging agent and as a carrier vehicle for targeted delivery of cytocidal agents to CD20-positive cancer cells.